摘要目的 检测幽门螺杆菌临床菌株groEL基因序列差异,确定重组表达的GroEL蛋白(r-GroEL)抗原性、免疫反应性以及对幽门螺杆菌感染小鼠的免疫保护性.方法 采用PCR及测序法了解幽门螺杆菌临床菌株groEL基因序列.构建幽门螺杆菌groEL基因pET42a-E.coli BL21DE3原核表达系统,采用SDS-PAGE及凝胶图像分析系统确定rGroEL表达量,Ni-NTA亲和层析法提纯rGroEL.采用ELISA和Western blot检测rGroEL的抗原性和免疫反应性,采用幽门螺杆菌全菌为包被抗原的ELISA确定GroEL膜定位.采用幽门螺杆菌小鼠感染模型了解rGroEL免疫保护作用.结果 35株幽门螺杆菌临床菌株groEL基因序列相似性高达99.4％～ 100％.rGroEL表达量可达细菌总蛋白的56％,SDS-PAGE后提纯的rGroEL在凝胶中显示为单一的条带.rGroEL可诱导家兔和小鼠产生高效价IgG抗体,同时也能被幽门螺杆菌全菌抗体(Hp-IgG)或rGroEL-IgG识别并与之结合.GroEL是幽门螺杆菌表面蛋白抗原.50、100或200 μg rGroEL免疫可分别使50.0％ (6/12)、75.0％ (9/12)和91.7％ (11/12)小鼠免于幽门 螺杆菌SS1株的感染,200 μg rGroEL免疫保护率(91.7％)显著高于50μg rGroEL(50.0％)( P＜0.05).结论 幽门螺杆菌GroEL蛋白是序列保守、具有良好免疫原性和免疫保护性的表面抗原,可作为基因工程疫苗候选抗原.

abstractsObjective To determine the sequence diversity in groEL gene of Helicobacter pylori isolates,and the antigenicity,immunoreactivity and immunoprotection of recombinant expressed GroEL (rGroEL) against H.pylori-infection in mice.Methods The sequences of groEL genes from H.pylori isolates were detected by PCR and sequencing.A prokaryotic expression system of H.pylori groEL gene using pET42a and E.coli BL21DE3 was generated.SDS-PAGE and Gel Image Analyzer were used to measure the yield of rGroEL and Ni-NTA affinity chromatography was applied to extract the expressed rGroEL.The antigenicity and immunoreactivity of rGroEL were examined using ELISA and Western blot assay.The membrane location of GroEL was determined by ELISA using whole cells of H.pylorias the coated antigen.The immunoprotection of rGroEL was detected in H.pylori-infected mouse model.Results All the 35 tested H.pylori isolates possess the groEL gene with 99.4％ to 100％ sequence identities.The yield of rGroEL expression occupied 56％ of the total bacterial proteins and the extracted rGroEL showed a single band in gel after SDSPAGE.rGroEL could induce the production of high-titer IgG in rabbits or mice.The lgG against whole cell of H.pylori(Hp-IgG) and rGroEL-IgG could recognize as well as combine with rGroEL protein.GroEL is a superficial protein antigen of H.pylori.The immunization with 50,100 or 200 μg rGroEL prevented 50.0％ (6/12),75.0％ (9/12) or 91.7％ (11/12) mice from infection of H.pylori strain SS1.The immunoprotective rate ( 91.7％ ) due to the immunization of 200 μg rG roEL was significantly higher than that ( 50.0％ ) of 50 μg rGroEL ( P＜0.05 ).Conclusion The GroEL protein is a sequence-conserved,immunogenic and immunoprotective superficial antigen of H.pylori,which can be used as the immunogen candidate for developing genetically engineering vaccines.